Research reportGap junctions in the chicken pineal gland
Introduction
Gap junctions are membrane specializations that contain intercellular channels permeable to ions and small molecules allowing for synchronization of the cellular responses of heterogeneous cells. Gap junctional channels are made of a family of related proteins called connexins (Cx; reviewed in Ref. [26]). Previous studies have demonstrated the presence of gap junctions in the pineal gland of several mammalian species 11, 18, 28, 29, 34, 39, 40, 50, 56, 58, 61and teleosts [46]. Molecular characterization of pineal gap junctions has been carried out only in the rat 3, 51, where functional coupling has been demonstrated [51]. Functional coupling has also been demonstrated in the fish pineal gland [19].
Similar to other species, the chicken pineal gland secretes melatonin with a circadian rhythm. Unlike the mammalian pineal gland, the chicken pineal gland is considered a photoreceptive organ. Studies in cultures of pineal cells from 1-day-old chickens, which maintain their ability to secrete melatonin with a circadian rhythm entrained by light [15], have been widely used to elucidate components and regulation of the biological clock that governs the neuroendocrine function of the gland. These studies have revealed that individual pinealocytes are photoreceptive, and that the gland behaves as an independent oscillator 43, 55. A fine control of transcription/translation of several target genes (e.g., kinases, ion channels and regulatory elements) through circadian oscillation in the activation of transcription factors underlies the circadian rhythm of melatonin release (reviewed in Refs. 24, 33). Moreover, in the suprachiasmatic nucleus of the rat, a mammalian oscillator, astrocyte gap junctions have been proposed to facilitate synchronization of oscillation of neuronal activity [59]. Although gap junctions might play a similar role in the chicken pineal gland, these membrane specializations have not been described among the cells of this tissue.
The chicken pineal gland is composed of follicles surrounded by extracellular stroma; each follicle surrounds a small lumen. According to their ultrastructural morphology, two different cell types lie within the follicle: pineal receptor cells (B pinealocytes) and astrocytes (ependymal cells or A pinealocytes), often forming two discrete epithelial layers surrounding the lumen 6, 9. We will refer to the former cells as pinealocytes. Most cells within a follicle are elongated and stretch between the lumen at their apical pole and the basal lamina at their basal pole. Parafollicular regions contain the same cell types, perhaps in less mature forms, in less organized fashion, sometimes surrounding narrow extracellular cavities. The interfollicular stroma contains various cell types, blood vessels, lymphoid tissue, fibroblastic cells and unmyelinated nerve fibers. Numerous junctional complexes containing zonula adherens have been found in the follicular and parafollicular zones throughout development 6, 9. Neuron cell bodies are rare or absent in the chicken pineal gland, although many neurites may invade the gland from sympathetic nerves 6, 12, 62.
The circadian rhythm of melatonin synthesis and release in the pineal organ may be enhanced by synchronization of the metabolic activity of individual pineal cells, because each pinealocyte is considered an independent oscillator 43, 55. Gap junctions are good candidates for synchronizing the response of heterogeneous cell types [21]. Given that the different cell types can be distinguished morphologically much better in the pineal gland of the chicken, this organism was chosen to address the existence of coupling between homologous and heterologous cells in a system that presents a circadian rhythm in its neuroendocrine function. Thus, the experiments presented here were designed to study the presence and localization of gap junctions in the chicken pineal gland, to identify molecular components of gap junctions, and to test for intercellular gap junctional communication within the gland.
Section snippets
Animals
White Leghorn chickens were obtained from SPAFAS (Norwich, CT). The animals were decapitated and the pineal gland was carefully removed from the cranium; special care was taken to minimize pain or discomfort.
Freeze fracture
The chicken pineal gland (1-day-old) was dissected and fixed in 2% buffered glutaraldehyde for 2 h, washed in buffer, and cryoprotected in 30% glycerol. The gland was then held between two gold discs and plunge frozen in liquid isopentane chilled by liquid nitrogen. The frozen gland was
Ultrastructure of gap junctions in the pineal gland
The presence of gap junctions in the chicken pineal gland was examined by freeze fracture. In freeze fracture replicas, small tight junctions and gap junctions were found in close association in many cases (Fig. 1B). More rarely, small gap junction arrays were found in isolation (Fig. 1A). Some extensive tight junction arrays were noted, which did not include obvious gap junctions (Fig. 1C), or where possible gap junction particles were ambiguous (Fig. 1D). Some of these longer tight junction
Discussion
Although the existence of numerous junctional complexes between cells of the chicken pineal gland had been previously described 6, 9, the present report is the first demonstration of gap junctions between these cells. Because of their very small size and dispersion, it is not surprising that gap junctions were previously missed. In addition, in freeze fracture replicas and in thin sections (Fig. 1, Fig. 2, Fig. 3, Fig. 4), tight junctions were observed in close relation to gap junctions. Our
Acknowledgements
This work was supported in part by NIH grants NS07512 (to M.V.L. Bennett), EY08368 and HD09402 (to E.C.B.) and FONDECYT grant 1990146 (to J.C.S.).
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